The regulation of smooth muscle and nonmuscle myosin by phosphorylation of the 20 kDa regulatory light chain (RLC) is of profound importance to contraction of smooth muscle tissue and the function of myosin in nonmuscle cells. The site of phosphorylation is in the neck region of myosin and is quite remote from the ATP and actin binding sites on myosin. The phosphorylation site lies within an area now referred to as the regulatory domain of myosin which consists of an alpha-helical stretch of heavy chain which forms the binding sites for the essential light chain (ELC) and RLC. The heavy chain dimerizes with itself just carboxyl-terminal to the RLC binding site and begins to form the rod portion. Thus, at this neck region, there are six polypeptide chains in close proximity and it is possible that interaction between the two necks might be important for regulation. We provide evidence for this by demonstrating that single-headed myosin prepared by proteolytically removing one of the two heads is no longer regulated by phosphorylation, but rather is constitutively active. Another aspect of the regulation of smooth and nonmuscle myosins is their ability to adopt a folded structure with a sedimentation coefficient of 10S. Here, the tail region of myosin loops back upon itself and appears to make contact with the regulatory domain region. The ability to form the 10S conformation is regulated by phosphorylation and is thought to be involved in the regulation of filament assembly and maybe a mechanism for further suppressing the enzymatic activity of unphosphorylated smooth muscle myosin. We cross-linked myosin while in the 10S conformation. A covalent cross-link was formed between Cys-108 of the RLC and the portion of the myosin heavy chain tail that contacts the regulatory domain during folding. The cross-linked myosin could not unfold when phosphorylated or when exposed to high ionic strength conditions. Our results show that the cross-linked, folded myosin is inactive even when the RLCs are phosphorylated.